Research in our laboratory focuses on the mechanisms of insulin resistance in human skeletal muscle. Specifically, we ask how serine/threonine phosphorylation of the signaling protein IRS-1 modulates the function of this protein, which is a key to insulin action in muscle. Using sensitive mass spectrometry techniques, we identified 22 serine/threonine phosphorylation sites in IRS-1 isolated from human vastus lateralis muscle. We quantified the effect of insulin on the in vivo phosphorylation of 15 of these residues. Of these 15 sites, phosphorylation of 4 residues, Ser348, Thr446, Thr495, and Ser1005, was decreased upon insulin treatment. The purpose of this proposal is to provide support for Dr. Langlais to ascertain whether the insulin controlled pattern and level and of phosphorylation of Ser348, Thr446, Thr495, and Ser1005 in human IRS-1 is deregulated in obese and type 2 diabetic human subjects as compared to lean, healthy controls. The second aim of this proposal is to identify the kinase(s) responsible for their phosphorylation, with mTOR being the principal candidate kinase. These experiments will provide a conceptual framework in which to place into context other results from our laboratory regarding regulation of phosphorylation of these sites in human muscle in vivo. Therefore Dr. Langlais' experiments will interweave with the larger mission of the laboratory. Definition of the molecular mechanisms responsible for insulin resistance will provide knowledge regarding the pathogenesis of type 2 diabetes mellitus and potentially provide molecular targets for treatment modalities. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE: Understanding how insulin fails to function properly in its target tissues, such as skeletal muscle, will provide insight into how individuals develop insulin resistance, type 2 diabetes, obesity, and the metabolic syndrome. Since these diseases are a growing public health problem responsible together for about 40% of public health costs, knowledge of the origins prevention and potential treatments for these diseases are imperative. [unreadable] [unreadable] [unreadable]